The tumor suppressor FOXO3a mediates the response to EGFR inhibition in glioblastoma cells

Cell Oncol (Dordr). 2019 Aug;42(4):521-536. doi: 10.1007/s13402-019-00443-1. Epub 2019 Apr 13.

Abstract

Purpose: Although EGFR activation is a hallmark of glioblastoma (GBM), anti-EGFR therapy has so far not yielded the desired effects. Targeting PI3K/Akt has been proposed as a strategy to increase the cellular sensitivity to EGFR inhibitors. Here we evaluated the contribution of FOXO3a, a key Akt target, in the response of GBM cells to EGFR inhibition.

Methods: FOXO3a activation was assessed by immunofluorescence and gene reporter assays, and by evaluating target gene expression using Western blotting and qRT-PCR. Cellular effects were evaluated using cell viability and apoptosis assays, i.e., Annexin V/PI staining and caspase 3/7 activity measurements. Drug synergism was evaluated by performing isobolographic analyses. Gene silencing experiments were performed using stable shRNA transfections.

Results: We found that EGFR inhibition in GBM cells led to FOXO3a activation and to transcriptional modulation of its key targets, including repression of the oncogene FOXM1. In addition, we found that specific FOXO3a activation recapitulated the molecular effects of EGFR inhibition, and that the FOXO3a activator trifluoperazine, a FDA-approved antipsychotic agent, reduced GBM cell growth. Subsequent isobolographic analyses of combination experiments indicated that trifluoperazine and erlotinib cooperated synergistically and that their concomitant treatment induced a robust activation of FOXO3a, leading to apoptosis in GBM cells. Using gene silencing, we found that FOXO3a is essential for the response of GBM cells to EGFR inhibition.

Conclusions: Our data indicate that FOXO3a activation is a crucial event in the response of GBM cells to EGFR inhibition, suggesting that FOXO3a may serve as an actionable therapeutic target that can be modulated using FDA-approved drugs.

Keywords: EGFR; Erlotinib; FOXO3a; Glioblastoma; Phenotiazines; Trifluoperazine.

MeSH terms

  • Brain Neoplasms / drug therapy
  • Brain Neoplasms / genetics
  • Brain Neoplasms / metabolism*
  • Brain Neoplasms / pathology
  • Cell Line, Tumor
  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism
  • Cell Proliferation / drug effects
  • ErbB Receptors / antagonists & inhibitors*
  • ErbB Receptors / metabolism
  • Erlotinib Hydrochloride / pharmacology
  • Forkhead Box Protein O3 / metabolism*
  • Glioblastoma / drug therapy
  • Glioblastoma / genetics
  • Glioblastoma / metabolism*
  • Glioblastoma / pathology
  • Humans
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinase Inhibitors / therapeutic use
  • Transcriptional Activation / genetics
  • Trifluoperazine / pharmacology
  • Trifluoperazine / therapeutic use

Substances

  • FOXO3 protein, human
  • Forkhead Box Protein O3
  • Protein Kinase Inhibitors
  • Trifluoperazine
  • Erlotinib Hydrochloride
  • ErbB Receptors